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INTRODUCTION: Tumors can be targeted by modulating the immune response of the patient. Programmed cell death protein 1 (PD-1) and programmed cell death ligand 1 (PD-L1) are critical immune checkpoints in cancer biology. The efficacy of certain cancer immunotherapies has been achieved by targeting these molecules using monoclonal antibodies. METHOD: Small-molecule drugs have also been developed as inhibitors of the PD-1/PD-L1 axis, with a mechanism of action that is distinct from that of antibodies: they induce the formation of PD-L1 homodimers, causing their stabilization, internalization, and subsequent degradation. Drug repurposing is a strategy in which new uses are sought after for approved drugs, expediting their clinical translation based on updated findings. In this study, we generated a pharmacophore model that was based on reported small molecules that targeted PD-L1 and used it to identify potential PD-L1 inhibitors among FDA-approved drugs. RESULTS: We identified 12 pharmacophore-matching compounds, but only 4 reproduced the binding mode of the reference inhibitors in docking experiments. Further characterization by molecular dynamics showed that pranlukast, an antagonist of leukotriene receptors that is used to treat asthma, generated stable and energyfavorable interactions with PD-L1 homodimers and induced homodimerization of recombinant PD-L1. CONCLUSION: Our results suggest that pranlukast inhibits the PD-1/PD-L1 axis, meriting its repurposing as an antitumor drug.

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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main Protease (Mpro) is an enzyme that cleaves viral polyproteins translated from the viral genome and is critical for viral replication. Mpro is a target for anti-SARS-CoV-2 drug development, and multiple Mpro crystals complexed with competitive inhibitors have been reported. In this study, we aimed to develop an Mpro consensus pharmacophore as a tool to expand the search for inhibitors. We generated a consensus model by aligning and summarizing pharmacophoric points from 152 bioactive conformers of SARS-CoV-2 Mpro inhibitors. Validation against a library of conformers from a subset of ligands showed that our model retrieved poses that reproduced the crystal-binding mode in 77% of the cases. Using models derived from a consensus pharmacophore, we screened >340 million compounds. Pharmacophore-matching and chemoinformatics analyses identified new potential Mpro inhibitors. The candidate compounds were chemically dissimilar to the reference set, and among them, demonstrating the relevance of our model. We evaluated the effect of 16 candidates on Mpro enzymatic activity finding that seven have inhibitory activity. Three compounds (1, 4, and 5) had IC50 values in the midmicromolar range. The Mpro consensus pharmacophore reported herein can be used to identify compounds with improved activity and novel chemical scaffolds against Mpro. The method developed for its generation is provided as an open-access code (https://github.com/AngelRuizMoreno/ConcensusPharmacophore) and can be applied to other pharmacological targets.

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SARS-CoV-2 Main Protease (Mpro) is an enzyme that cleaves viral polyproteins translated from the viral genome, which is critical for viral replication. Mpro is a target for anti-SARS-CoV-2 drug development. Herein, we performed a large-scale virtual screening by comparing multiple structural descriptors of reference molecules with reported anti-coronavirus activity against a library with >17 million compounds. Further filtering, performed by applying two machine learning algorithms, identified eighteen computational hits as anti-SARS-CoV-2 compounds with high structural diversity and drug-like properties. The activities of twelve compounds on Mpro's enzymatic activity were evaluated by fluorescence resonance energy transfer (FRET) assays. Compound 13 (ZINC13878776) significantly inhibited SARS-CoV-2 Mpro activity and was employed as a reference for an experimentally hit expansion. The structural analogues 13a (ZINC4248385), 13b (ZNC13523222), and 13c (ZINC4248365) were tested as Mpro inhibitors, reducing the enzymatic activity of recombinant Mpro with potency as follows: 13c > 13 > 13b > 13a. Then, their anti-SARS-CoV-2 activities were evaluated in plaque reduction assays using Vero CCL81 cells. Subtoxic concentrations of compounds 13a, 13c, and 13b displayed in vitro antiviral activity with IC50 in the mid micromolar range. Compounds 13a-c could become lead compounds for the development of new Mpro inhibitors with improved activity against anti-SARS-CoV-2.

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Autism spectrum disorder (ASD) is a diverse group of neurodevelopmental conditions with complex origins. Individuals with ASD present various neurobiological abnormalities, including an altered immune response in the central nervous system and other tissues. Animal models like the C58/J inbred mouse strain are used to study biological characteristics of ASD. This strain is considered an idiopathic autism model because of its demonstrated reduced social preference and repetitive behaviours. Notably, C58/J mice exhibit alterations in dendritic arbour complexity, density and dendritic spines maturation in the hippocampus and prefrontal cortex (PFC), but inflammatory-related changes have not been explored in these mice. In this study, we investigated proinflammatory markers in the hippocampus and PFC of adult male C58/J mice. We discovered elevated levels of interferon gamma (IFN-γ) and monocyte chemoattractant protein 1 (MCP-1) in the hippocampus, suggesting increased inflammation, alongside a reduction in the anti-inflammatory enzyme arginase 1 (ARG1). Conversely, the PFC displayed reduced levels of TNF-α and MCP-1. Microglial analysis revealed higher levels of transmembrane protein 119 (TMEM119) and increased microglial density in a region-specific manner of the autistic-like mice, particularly in the PFC and hippocampus. Additionally, an augmented expression of the fractalkine receptor CX3CR1 was observed in the hippocampus and PFC of C58/J mice. Microglial morphological analysis shows no evident changes in the hippocampus of mice with autistic-like behaviours versus wild-type strain. These region-specific changes can contribute to modulate processes like inflammation or synaptic pruning in the C58/J mouse model of idiopathic autism.

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COVID-19 patients can exhibit a wide range of clinical manifestations affecting various organs and systems. Neurological symptoms have been reported in COVID-19 patients, both during the acute phase of the illness and in cases of long-term COVID. Moderate symptoms include ageusia, anosmia, altered mental status, and cognitive impairment, and in more severe cases can manifest as ischemic cerebrovascular disease and encephalitis. In this narrative review, we delve into the reported neurological symptoms associated with COVID-19, as well as the underlying mechanisms contributing to them. These mechanisms include direct damage to neurons, inflammation, oxidative stress, and protein misfolding. We further investigate the potential of small molecules from natural products to offer neuroprotection in models of neurodegenerative diseases. Through our analysis, we discovered that flavonoids, alkaloids, terpenoids, and other natural compounds exhibit neuroprotective effects by modulating signaling pathways known to be impacted by COVID-19. Some of these compounds also directly target SARS-CoV-2 viral replication. Therefore, molecules of natural origin show promise as potential agents to prevent or mitigate nervous system damage in COVID-19 patients. Further research and the evaluation of different stages of the disease are warranted to explore their potential benefits.

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Anthocyanins are a type of flavonoids that give plants and fruits their vibrant colors. They are known for their potent antioxidant properties and have been linked to various health benefits. Upon consumption, anthocyanins are quickly absorbed and can penetrate the blood-brain barrier (BBB). Research based on population studies suggests that including anthocyanin-rich sources in the diet lower the risk of neurodegenerative diseases. Anthocyanins exhibit neuroprotective effects that could potentially alleviate symptoms associated with such diseases. In this review, we compiled and discussed a large body of evidence supporting the neuroprotective role of anthocyanins. Our examination encompasses human studies, animal models, and cell cultures. We delve into the connection between anthocyanin bioactivities and the mechanisms underlying neurodegeneration. Our findings highlight how anthocyanins' antioxidant, anti-inflammatory, and anti-apoptotic properties contribute to their neuroprotective effects. These effects are particularly relevant to key signaling pathways implicated in the development of Alzheimer's and Parkinson's diseases. In conclusion, the outcome of this review suggests that integrating anthocyanin-rich foods into human diets could potentially serve as a therapeutic approach for neurological conditions, and we identify promising avenues for further exploration in this area.

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CD36 is highly expressed in diverse tumor types and its expression correlates with advanced stages, poor prognosis, and reduced survival. In cancer cells, CD36: 1) increases fatty acid uptake, reprogramming lipid metabolism; 2) favors cancer cell proliferation, and 3) promotes epithelial-mesenchymal transition. Furthermore, CD36 expression correlates with the expression of cancer stem cell markers and CD36+ cancer cells display increased stemness functional properties, including clonogenicity, chemo- and radioresistance, and metastasis-initiating capability, suggesting CD36 is a marker of the cancer stem cell population. Thus, CD36 has been pointed as a potential therapeutic target in cancer. At present, at least three different types of molecules have been developed for reducing CD36-mediated functions: blocking monoclonal antibodies, small-molecule inhibitors, and compounds that knock-down CD36 expression. Herein, we review the role of CD36 in cancer progression, its participation in stemness control, as well as the efficacy of reported CD36 inhibitors in cancer cell cultures and animal models. Overall, the evidence compiled points that CD36 is a valid target for the development of new anti-cancer therapies.

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Therapeutic monoclonal antibodies (mAbs) are complex bioengineered proteins that require to be routinely characterized with robust and reliable bioassays. Infliximab was the first anti-TNFα mAb approved for use in humans and its use has revolutionized the treatment TNF-mediated inflammatory disorders. The mechanism of action (MOA) of infliximab involves its binding to soluble (s) and membrane (m) TNFα. Here, we describe two simple in vitro bioassays for the assessment of key activities of infliximab. First, a bioassay for TNFα neutralization, which evaluates the Fab binding to sTNFα and the consequent reduction in the activation of TNFα receptors and TNFα-induced expression of adhesion molecules on endothelial cells. A second bioassay evaluates the triggering of Complement-Dependent Cytotoxicity (CDC) in cells expressing mTNFα, which requires the interaction of infliximab-Fc with proteins of the complement system. In both cases, the biological responses are measured by flow cytometry, which is accessible for most laboratories. The methods reported here can be easily adapted to other therapeutic mAbs with similar MOA.

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Cancer stem cells (CSCs) are tumor cells that share functional characteristics with normal and embryonic stem cells. CSCs have increased tumor-initiating capacity and metastatic potential and lower sensitivity to chemo- and radiotherapy, with important roles in tumor progression and the response to therapy. Thus, a current goal of cancer research is to eliminate CSCs, necessitating an adequate phenotypic and functional characterization of CSCs. Strategies have been developed to identify, enrich, and track CSCs, many of which distinguish CSCs by evaluating the expression of surface markers, the initiation of specific signaling pathways, and the activation of master transcription factors that control stemness in normal cells. We review and discuss the use of reporter gene systems for identifying CSCs. Reporters that are under the control of aldehyde dehydrogenase 1A1, CD133, Notch, Nanog homeobox, Sex-determining region Y-box 2, and POU class 5 homeobox can be used to identify CSCs in many tumor types, track cells in real time, and screen for drugs. Thus, reporter gene systems, in combination with in vitro and in vivo functional assays, can assess changes in the CSCs pool. We present relevant examples of these systems in the evaluation of experimental CSCs-targeting therapeutics, demonstrating their value in CSCs research.

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CD44 promotes metastasis, chemoresistance, and stemness in different types of cancer and is a target for the development of new anti-cancer therapies. All CD44 isoforms share a common N-terminal domain that binds to hyaluronic acid (HA). Herein, we used a computational approach to design new potential CD44 antagonists and evaluate their target-binding ability. By analyzing 30 crystal structures of the HA-binding domain (CD44HAbd), we characterized a subdomain that binds to 1,2,3,4-tetrahydroisoquinoline (THQ)-containing compounds and is adjacent to residues essential for HA interaction. By computational combinatorial chemistry (CCC), we designed 168,190 molecules and compared their conformers to a pharmacophore containing the key features of the crystallographic THQ binding mode. Approximately 0.01% of the compounds matched the pharmacophore and were analyzed by computational docking and molecular dynamics (MD). We identified two compounds, Can125 and Can159, that bound to human CD44HAbd (hCD44HAbd) in explicit-solvent MD simulations and therefore may elicit CD44 blockage. These compounds can be easily synthesized by multicomponent reactions for activity testing and their binding mode, reported here, could be helpful in the design of more potent CD44 antagonists.

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ABSTRACT Programmed cell death protein 1 (PD-1) and its ligand, programmed death-ligand-1 (PD-L1), play key roles in the suppression of the cytotoxic activity of T cells. PD-L1 is overexpressed on various types of cancer cells, leading to immune evasion. In the past decade, therapeutic antibodies that target the PD-1/PD-L1 axis have been developed to inhibit the immune suppression triggered by these two proteins. At present, five antibodies (two anti-PD-1 and three anti-PD-L1) have received approval by regulatory agencies in the US and Europe. In this work, we aimed to review their clinical applications and adverse effects. Furthermore, using their reported crystal structures, we discuss the similarities and differences between the PD-1/PD-L1 interface and the epitopes that are recognized by the antibodies. Detailed analyses of the contact residues involved in the ligand-receptor and target-antibody interactions have shown partial overlap. Altogether, the data presented here demonstrate that: (1) in contrast to other therapeutic antibodies, anti-PD-1/PD-L1 has a wide range of clinical applications; (2) these targeted therapies are not exempt from adverse effects; and (3) the characterization of the structural domains that are recognized by the antibodies can guide the development of new PD-1- and PD-L1-blocking agents. (REV INVEST CLIN. 2021;73(1):8-16)

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CK1ε is a key regulator of WNT/ß-catenin and other pathways that are linked to tumor progression; thus, CK1ε is considered a target for the development of antineoplastic therapies. In this study, we performed a virtual screening to search for potential CK1ε inhibitors. First, we characterized the dynamic noncovalent interactions profiles for a set of reported CK1ε inhibitors to generate a pharmacophore model, which was used to identify new potential inhibitors among FDA-approved drugs. We found that etravirine and abacavir, two drugs that are approved for HIV infections, can be repurposed as CK1ε inhibitors. The interaction of these drugs with CK1ε was further examined by molecular docking and molecular dynamics. Etravirine and abacavir formed stable complexes with the target, emulating the binding behavior of known inhibitors. However, only etravirine showed high theoretical binding affinity to CK1ε. Our findings provide a new pharmacophore for targeting CK1ε and implicate etravirine as a CK1ε inhibitor and antineoplastic agent.

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Molecular docking is a useful and powerful computational method for the identification of potential interactions between small molecules and pharmacological targets. In reverse docking, the ability of one or a few compounds to bind a large dataset of proteins is evaluated in silico. This strategy is useful for identifying molecular targets of orphan bioactive compounds, proposing new molecular mechanisms, finding alternative indications of drugs, or predicting drug toxicity. Herein, we describe a detailed reverse docking protocol for the identification of potential targets for 4-hydroxycoumarin (4-HC). Our results showed that RAC1 is a target of 4-HC, which partially explains the biological activities of 4-HC on cancer cells. The strategy reported here can be easily applied to other compounds and protein datasets.

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BACKGROUND: The deterioration of the skin accentuates over time, affecting its aesthetic appearance. This is characterized by the weakening of the mechanisms involved in the regeneration and repair of the dermal matrix. Consequently, the skin losses elasticity and smoothness resulting in the formation of wrinkles. The alternatives for facial rejuvenation include surgery, injection of botulinum toxin, and the application of masks. Topic products are less invasive, can be self-applied, and have an increased benefit/risk relationship. AIM: We developed a liquid formulation containing collagen hydrolyzed and evaluated the product by cutting-edge technology in order to define proper its quality attributes. METHODS: We employed nuclear magnetic resonance (NMR), size-exclusion chromatography (SEC), and mass spectrometry (MS). Additionally, we analyzed its cosmetical effect in five volunteers and we demonstrate the product safety. RESULTS: Our results demonstrate the following: (a) a stable secondary structure identity associated to the known triple helix arrangement in liquid and solid states; (b) a typical conformational flexibility depending on its hydration state; (c) thermal stability confirmed by liquid- and solid-state nuclear magnetic resonance schemes; and (d) a molecular mass distribution of peptides between 0.5 and 19.5 kDa. The product faded wrinkles in the forehead, an effect that remained after removing the mask. The formula was non-irritating and hypoallergenic. CONCLUSION: We characterized, using state-of-the-art methodologies, the quality attributes that are critical for the safety and beneficial effect of a new collagen-containing formula.

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Programmed cell death protein 1 (PD-1) and its ligand, programmed death-ligand-1 (PD-L1), play key roles in the suppression of the cytotoxic activity of T cells. PD-L1 is overexpressed on various types of cancer cells, leading to immune evasion. In the past decade, therapeutic antibodies that target the PD-1/PD-L1 axis have been developed to inhibit the immune suppression triggered by these two proteins. At present, five antibodies (two anti-PD-1 and three anti-PD-L1) have received approval by regulatory agencies in the US and Europe. In this work, we aimed to review their clinical applications and adverse effects. Furthermore, using their reported crystal structures, we discuss the similarities and differences between the PD-1/PD-L1 interface and the epitopes that are recognized by the antibodies. Detailed analyses of the contact residues involved in the ligand-receptor and target-antibody interactions have shown partial overlap. Altogether, the data presented here demonstrate that: (1) in contrast to other therapeutic antibodies, anti-PD-1/PD-L1 has a wide range of clinical applications; (2) these targeted therapies are not exempt from adverse effects; and (3) the characterization of the structural domains that are recognized by the antibodies can guide the development of new PD-1- and PD-L1-blocking agents.

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INTRODUCTION: Cancer stem cells (CSCs) drive the initiation, maintenance, and therapy response of breast tumors. CD49f is expressed in breast CSCs and functions in the maintenance of stemness. Thus, blockade of CD49f is a potential therapeutic approach for targeting breast CSCs. In the present study, we aimed to repurpose drugs as CD49f antagonists. MATERIALS AND METHODS: We performed consensus molecular docking using a subdomain of CD49f that is critical for heterodimerization and a collection of pharmochemicals clinically tested. Molecular dynamics simulations were employed to further characterize drug-target binding. Using MDA-MB-231 cells, we evaluated the effects of potential CD49f antagonists on 1) cell adhesion to laminin; 2) mammosphere formation; and 3) cell viability. We analyzed the effects of the drug with better CSC-selectivity on the activation of CD49f-downstream signaling by Western blot (WB) and co-immunoprecipitation. Expressions of the stem cell markers CD44 and SOX2 were analyzed by flow cytometry and WB, respectively. Transactivation of SOX2 promoter was evaluated by luciferase reporter assays. Changes in the number of CSCs were assessed by limiting-dilution xenotransplantation. RESULTS: Pranlukast, a drug used to treat asthma, bound to CD49f in silico and inhibited the adhesion of CD49f+ MDA-MB-231 cells to laminin, indicating that it antagonizes CD49f-containing integrins. Molecular dynamics analysis showed that pranlukast binding induces conformational changes in CD49f that affect its interaction with ß1-integrin subunit and constrained the conformational dynamics of the heterodimer. Pranlukast decreased the clonogenicity of breast cancer cells on mammosphere formation assay but had no impact on the viability of bulk tumor cells. Brief exposure of MDA-MB-231 cells to pranlukast altered CD49f-dependent signaling, reducing focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K) activation. Further, pranlukast-treated cells showed decreased CD44 and SOX2 expression, SOX2 promoter transactivation, and in vivo tumorigenicity, supporting that this drug reduces the frequency of CSC. CONCLUSION: Our results support the function of pranlukast as a CD49f antagonist that reduces the CSC population in triple-negative breast cancer cells. The pharmacokinetics and toxicology of this drug have already been established, rendering a potential adjuvant therapy for breast cancer patients.

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Therapeutic antibodies are recombinant proteins used in the treatment of cancer. There is a new generation of monoclonal antibodies with activity against cancer cells, known as antibody-drug conjugates. These molecules are made up of three elements: a monoclonal antibody, a highly potent cytotoxic drug, and a chemical linker that binds them together. The antibody recognizes tumor antigens, thereby allowing targeted delivery of the cytotoxic agent to cancer cells. After recognizing its antigen, the antibody-drug conjugate is endocytosed by the target cells, where the protein fraction is degraded into lysosomes, releasing the cytotoxic drug. This article reviews antibody-drug conjugates general characteristics and describes the clinical evidence of efficacy and safety of the first four approved by regulatory agencies in the United States and Europe.

Los anticuerpos terapéuticos son proteínas recombinantes empleadas en el tratamiento del cáncer. Existe una nueva ­generación de anticuerpos monoclonales con actividad contra las células cancerosas, conocidos como anticuerpos conjugados a fármacos. Estas moléculas están integradas por tres elementos: un anticuerpo monoclonal, un fármaco citotóxico con alta potencia y un enlazador químico que los une. El anticuerpo reconoce antígenos tumorales, por lo que permite la entrega dirigida del agente citotóxico hacia las células cancerosas. Tras el reconocimiento de su antígeno, el anticuerpo conjugado a fármaco es endocitado por las células blanco, donde se induce la degradación lisosomal de la fracción proteica y se libera el fármaco citotóxico. En el presente artículo se revisan las características generales de los anticuerpos conjugados a fármacos y se describe la evidencia clínica de la eficacia y seguridad de los primeros cuatro aprobados por las agencias reguladoras de Estados Unidos y Europa.

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Resumen Los anticuerpos terapéuticos son proteínas recombinantes empleadas en el tratamiento del cáncer. Existe una nueva generación de anticuerpos monoclonales con actividad contra las células cancerosas, conocidos como anticuerpos conjugados a fármacos. Estas moléculas están integradas por tres elementos: un anticuerpo monoclonal, un fármaco citotóxico con alta potencia y un enlazador químico que los une. El anticuerpo reconoce antígenos tumorales, por lo que permite la entrega dirigida del agente citotóxico hacia las células cancerosas. Tras el reconocimiento de su antígeno, el anticuerpo conjugado a fármaco es endocitado por las células blanco, donde se induce la degradación lisosomal de la fracción proteica y se libera el fármaco citotóxico. En el presente artículo se revisan las características generales de los anticuerpos conjugados a fármacos y se describe la evidencia clínica de la eficacia y seguridad de los primeros cuatro aprobados por las agencias reguladoras de Estados Unidos y Europa.

Abstract Therapeutic antibodies are recombinant proteins used in the treatment of cancer. There is a new generation of monoclonal antibodies with activity against cancer cells, known as antibody-drug conjugates. These molecules are made up of three elements: a monoclonal antibody, a highly potent cytotoxic drug, and a chemical linker that binds them together. The antibody recognizes tumor antigens, thereby allowing targeted delivery of the cytotoxic agent to cancer cells. After recognizing its antigen, the antibody-drug conjugate is endocytosed by the target cells, where the protein fraction is degradated into lysosomes, releasing the cytotoxic drug. This article reviews antibody-drug conjugates general characteristics and describes the clinical evidence of efficacy and safety of the first four approved by regulatory agencies in the United States and Europe.

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Technological advances allowed the development of high-throughput instruments such as IntelliCyt iQue Screener PLUS®. Here, we took advantage of this technology to transfer a previously validated cytotoxicity assay. The evaluated parameters were cell permeability, caspase activation and phosphatidyl serine exposure. The assay was accurate (r2 = 0.90), precise (%CV ≤ 18.90) and specific. These results showed that this technology is suitable to be used in control quality environments. In addition, the automation provided a faster acquisition and analysis of data with precise and accurate results. This application could be implemented to evaluate another in vitro mechanism of action of different biotherapeutics.

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Identity is a critical quality attribute that must be determined before releasing batches of medicinal and dietary products. However, the identities of peptide-derived products composed of a large number of diverse molecules is challenging since most analytical techniques cannot analyze multiple molecules simultaneously. Here, we proposed the determination of the weight-average molecular weight (Mw) and polydispersity index (PDI) by mass spectrometry for control quality for the batch release of complex products, namely, glatiramer acetate (Copaxone), collagen hydrolysate (Colagenart), and a human dialyzable leucocyte extract (Transferon). The Mw and PDI values were orthogonally determined by PFG-STE-H2O(presaturation)-DOSY-NMR analysis. To the best of our knowledge, this is the first time that MS and NMR spectra have been combined to determine the PDI of complex products derived from protein hydrolysis that are not monodisperse. The performance of each method was evaluated by comparing the obtained results to those reported for glatiramer acetate using MALLS, the technique commonly employed to determine PDI. This combined approach demonstrates the ability of these techniques to separate peptide populations from complex mixtures to establish their identity through their mass distribution profiles.

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